المجلــــد (2) ، العـــــدد (6)،...Table (1) showed that the highest organogenesis percentage (100% ) was found on MS medium augmented with 0.5 and 1.0mgl-1 BA

0202يناير ( 5العدد ) – الثالثاجمللد اجمللة العربية للعلوم الزراعية

13 3

Organogenesis of Tomato (Lycopersicon

esculentum Mill.) cv. Sandra and Rocky by

Hypocotyl, Cotyledon and Leaf Explants

By

Gharbia Hormiz Danial - Diaa Ayoub Ibrahim Scientific Research Center (SRC), College of Science, University of Duhok,

Duhok, IRAQ

Doi: 10.21608/asajs.2020.67988 2102/ 00/ 21قبول النشر : 2102/ 01 / 2استالم البحث :

ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ

:المستخلص

l(Lycopersicon esculentumحكى اععاء صف ي باث انطاغت

Mill.) cv. Sandra and Rocky باسخخذاو االوساق انفهقت وانسقا ححج انفهقت

حذاخالث نخاض صف ي باث وقطع االوساق بخقت انضساعت انسجتيج

cv. Sandra and Rocky (.Lycopersicon esculentum Mill)انطاغت

باسخعال االوساق انفهقت وانسقاث ححج انفهقت فعال ع قطع االوساق. اخزث انقطع

26-23انباحت ي بخاث اسخحصهج بطشقت انضساعت انسجت كصذس نها بعش

انغزائ انذعى بخشاكض يخخهفت ي يظاث انى MSسػ ىو وصسعج عهى و

% نخىانذ االععاء ف انسق ححج انفهقت اناي ف 211انباحت . حى حسجم سبت

يهغى / نخش كاخ 1.6و BAيهغى ظ نخش بضام اد 2انذعى باظافت MSوسػ

5.14ساذسا و جضء خعشي / قطعت باحت ف انصف 6..4وانزي سجم حىان

%( 211نهصف سوك كعذل عاو . با سجم اعهى سبت حكى نالععاء )

يهغى / نخش 1.6يهغى / نخش بضام اد و 2.6باسخعال االوساق انفهقت باظافت

جضء خعشي / قطعت باحت ف انصف ساذسا. 4.6كاخ وانزي سجم يا قاسب

سبت نخكى االععاء كاج قذ نىحظج ف انىسػ وعهى انقط ي رنك فا اعهى

يهغى / نخش كاخ 2يهغى / نخش بضام اد و 3وانذعى باظافت MSانغزائ

أظهشث جضء خعشي / نكم قطعت باحت ااصف سوك . .4.2وبعذل قذ حشاوح

فثان يهغى / نخش( ي 2و 1.6، 1.3، 1.1خائج انخذاخالث ب انبضام اد و )

% ( .42.7حايط انخهك ا اعهى سبت نخىنذ االععاء ي انسقا ححج انفهقت )

جضء خعشي / 5% بعذل .77.7جضء خعشي / قطعت باحت و 7.62بعذل

انذعى MSقطعت باحت ف انصف ساذسا و سوك عهى انخىان وانايت ف وسػ

Gharbia Danial - Diaa Ibrahim

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13

%( 211با اعهى سبت حكى نالععاء ) يهغى / نخش بضام اد نىحذ 6باظافت

نهصف سوك ف 4.13نهصف ساذسا و .4.7ي االوساق انفهقت نكال انصف )

يهغى فثان حايط انخهك و 1.3يهغى بضام اد و 6انذعى باظافت MSوسػ

جضء خعشي / قطعت باحت ف .7. 77و 3....يهغى/ نخش كاخ ، 1.6

ساذسا وسوك عهى انخىان. قهج انباحاث انخاضة بجاح انى ظشوف انصف

انحقم نغشض اقهخها.Abstract:

In vitro regeneration protocols were established for two

Tomato (Lycopersicon esculentum Mill.) cultivars (Sandra and

Rocky) grown in Kurdistan region using hypocotyl,

cotyledonous leaves and leaf explants. The explants excised

from 12-15 days old of in vitro plantlets cultured on MS medium

supplemented with different concentrations and combinations of

plant growth regulators. High organogenesis percentage (100%)

was achieved from hypocotyl grown in MS medium augmented

with 1.0mgl-1

BA+ 0.5mgl-1

kinetin, which record approximately

3.75 in Sandra and 4.09 in Rocky cultivar as average number of

shoots produced from each explant. While the highest

organogenesis percentage (100%) was formed from cotyledon

leaves using 1.5 mgl-1

BA+0.5mgl-1

kinetin that gave around 3.5

in Sandra cultivar as average number of shoots / explant. In

contrast, the highest organogenesis was found on MS medium

supplemented with 2.0mgl-1

BA+1.0 mgl-1

kinetin and the

average number of shoots produced from each explant were

approximately 3.17 in Rocky cultivar. The results of

combination between 5.0mgl-1

BA with (0.0, 0.2, 0.5 and

1.0)mgl-1

NAA, indicated that the highest organogenesis

percentage was recorded from hypocotyl (91.67 % with 6.5

shoots/explant and 66.67% with 4.0 shoots/ explant) in Sandra

and Rocky cultivars respectively grown on MS medium

supplemented with 5.0 mgl-1

BA alone. While the highest

organogenesis percentage (100%) was induced from


11 3

cotyledonous leaves in both cultivars (3.67 shoots for Sandra and

3.08 for Rocky) on MS medium supplemented with 5.0mgl-1

BA

+0.2mgl-1

NAA. Meanwhile, the highest organogenesis

percentage was obtained from leaf explants for both cultivars on

the combination of 1.0mgl-1

BA +0.5mgl-1

kinetin, (77.78% and

66.67%) in Sandra and Rocky cultivars respectively. The

regenerated plants were successfully transplanted to the field for

acclimatization

Keywords Lycopersicon esculentum; Micropropagation;

Organogenesis; TDZ, Kinetin.

Introduction

Tomato (Lycopersicon esculentum Mill.) is an important

solanaceous crop, and is widely grown in the world. As a highly

valuable and nutritious food, tomato is one of the major

vegetables consumed by humans throughout the world. It is

considered as the second most important vegetable crop after

potato, being grown in tropical, sub-tropical and temperate areas

(Mohamed et al., 2010). Tomato plays a vital role in

maintaining human health and vigor. It has antioxidant

properties and it is a source of vitamin B, C and β-carotene, rich

in fiber. Moreover, tomato is cholesterol free, hence, this feature

gave it a medical significance to use for decrease the blood

cholesterol, blood pressure, regulate blood sugar and boosts the

immunity. Furthermore, consumption of tomatoes and its

products decrease the risk of prostate cancer. As well as, it is a

major component of vegetarian and non-vegetarian diets (Rao

and Agarwal, 2000; Soundararajan, 2015).

In plant tissue culture, the organogenesis is the

differentiation of unorganized mass of cells to development of

adventitious organs. This system is commonly produced in callus


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culture, but it can be produced directly from the explants. It is

regulated through a balance between the levels of auxins and

cytokinins (Mohammed and Omar 1990). Tomato direct

regeneration from hypocotyl and cotyledonous leaves has been

previously reported via organogenesis in several researches

using different levels of plant growth regulators (Abu-El-Heba et

al., 2008; Mohamed et al., 2010; Wayase and Shitole 2014;

Kalyani and Rao 2014; Soundararajan, 2015).

Many researches and reports showed a great deal of

interesting to develop tomato crop by introducing value

agronomic traits by either plant tissue culture or genetic

transformation (Frary and Van Eck, 2005; Kole et al., 2010;

Orozco-Cárdenas et al., 2014). Successful application of

biotechnology in tomato improvement and tomato genetic

transformation depends upon the type of explants and

availability of efficient plant regeneration protocol, these factors

could limits the plant genetic transformation (Evans, 1989). The

current study will bring us closer to our goal of improving the

tomato cultivars in Kurdistan Region of Iraq for both herbicide

and abiotic stress resistance. However, there is no report on

multiple shoots induction and direct regeneration from

hypocotyls and cotyledon explants in tomato cv. (‘Sandra’ and

‘Rocky’) the two tomato genotypes that commonly grown in

Kurdistan region of Iraq.

Materials and Methods

Seeds of tomato cultivars Sandra and Rocky were obtained

from (Agriculture research station) Duhok Province, Kurdistan

Region of Iraq in March, 2016. To remove dirt’s and dusts, the

seeds were thoroughly washed using running tap water, then the

seeds were surface sterilized by immersing them in a mixture of

5% sodium hypochlorite (NaOCl) solution with few drops of

Tween-20 surfactant for 15 minutes. The seeds were removed


13 3

and rinsed 3 times with autoclaved deionized water, blotted dry.

Culture vessels containing MS (Murashige and Skoog, 1962)

basal medium were used for culturing the sterilized seeds then

were incubated in the dark for three days, transferred to growth

room with a day photoperiod (16 hours light /8 hours dark at

25±2 °C. Twelve to fifteen days after seed germination,

hypocotyl, cotyledon and leaf explants were excised. Hypocotyl

was sliced into 1-cm explants and cotyledons were sliced into 1-

cm2. One group of those explants was cultured on MS sterilized

medium supplemented with (0.5, 1.0 mgl-1

) of Kinetin combined

with (0.5, 1.0, 1.5 and 2.0 mgl-1

) of BA. Other groups of explants

were cultured on MS medium supplemented with 5.0mgl-1

BA

combined with 0.0, 0.2, 0.5 and 1.0mgl-1

NAA, a total of 15

replicates were initiated for each treatment.

After six weeks of incubation, the organogenesis

percentage, number of shoots and average shoots length were

recorded.

Data Analysis

All the comparisons between means were carried out

according to Duncan's multiple range test (P< 0.05) using a

computerized program of SAS.

Results

The key objective of this study is to obtain in vitro plants

regeneration through organogenesis from hypocotyls,

cotyledonous leaves and leaf sections. Hence, this technique can

be used as a part of tomato genetic transformation protocol and

then can transfer these new techniques into Kurdistan Region, as

well as to encourage investment in this field of biotechnology for

developing agriculture in Kurdistan.

During the present study, cotyledon, hypocotyl and leaf

sections of two tomato genotypes (Sandra and Rocky) were


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excised and gained from in vitro grown seedlings (12-15 days

old) as a source (Fig.1,A).

1. Hypocotyl organogenesis

Hypocotyl explants were cultured on MS medium

supplemented with various combinations and concentrations of

plant growth regulators. Two weeks after culture incubation,

shoot induction was initiated. At the end of the 6 weeks of

culture incubation, several shoots were initiated at the ends of

the hypocotyl explants (Table1 and 2).

Table 1: Effect of different combinations between BA and

Kinetin on direct organogenesis from hypocotyl explants of

tomato (Lycopersicon esculentum Mill.) cv. Sandra and

Rocky after six weeks of culture incubation. Tomato

cultivars

Growth

regulators

Organogenesis

%

Shoots/

explant

average

Length

average

(cm)

Sandra

0.5mgl-1

BA+ 0.5

mgl-1

Kinetin

100 2.42 b 0.39 b

1. mgl-1

BA+

0.5 mgl-1

Kinetin

100 3.75 a 0.54 b

1.5 mgl-1

BA+ 0.5

mgl-1

Kinetin

91.67 2.17 b 0.54 b

2.0 mgl-1

BA+ 1.0

mgl-1

Kinetin

83.34 1.67 b 0.43 b

0.5 mgl-1

BA+ 0.5 100 3.09 a 1.13 a


13 3

Rocky

mgl-1

Kinetin

1.0 mgl-1

BA+ 0.5

mgl-1

Kinetin

100 4.09 a 1.15 a

1.5 mgl-1

BA+ 0.5

mgl-1

Kinetin

66.67 1.59 b 0.38 b

2.0 mgl-1

BA+ 1.0

mgl-1

Kinetin

91.67 3.42 a 0.35 b

Various letters in columns clarify significant differences at 5%

level according to Duncan test.

Table (1) showed that the highest organogenesis

percentage (100% ) was found on MS medium augmented with

0.5 and 1.0mgl-1

BA combined with 0.5 mgl-1

kinetin for both

cultivar Sandra (Fig.1,B) and Rocky (Fig.2,A), and the high

average number of shoots produced from each explant was

approximately 3.75 and 4 in Sandra and Rocky respectively.

While lower organogenesis percentage (66.67%) with lower

shoots number 1.59 /explants was recorded in Rocky cultivar on

MS medium content 1.5mgl-1

BA combined with 0.5mgl-1

kinetin. On the other hand, high shoots length 1.15 cm was

recorded in Rocky cultivar on MS medium supplemented with

1.0 mgl-1

BA+0.5mgl-1

kinetin that is significantly higher when

compared with other growth regulator concentrations.

To study the effect of cytokinin with or without auxin

combination on the organogenesis from hypocotyl explants, high

concentration of BA (5.0 mgl-1

) + different concentrations of


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NAA (0.0, 0.2, 0.5, 1.0 mgl-1

) were added to MS medium which

was used for hypocotyl explants regeneration (Table 2).

Table 2: Effects of different combinations between BA and

NAA on direct organogenesis from hypocotyl explants of


Rocky after six weeks of culture incubation.

Tomato

cultivar

Growth

regulators

Organogenesis

%

Shoots/

explant

average

Length

average

(cm)

Sandra

5.0 mgl-1

BA+ 0.0

mgl-1

NAA

91.67 a 6.5 a 0.88 a

5.0 mgl-

1BA+ 0.2

mgl-1

NAA

0 c 0 c 0 c

5.0 mgl-1

BA+ 0.5

mgl-1

NAA

0 c 0 c 0 c

5.0 mgl-1

BA+ 1.0

mgl-1

NAA

0 c 0 c 0 c

Rocky

5.0mgl-1

BA+

0.0 mgl-1

NAA

66.67 b 4.0 b 0.5 b

5.0 mgl-

1BA+ 0.2

mgl-1

NAA

8.33 c 0.25 c 0.25 b

5.0 mgl-1

BA+ 0.5

mgl-1

NAA

0 c 0 c 0 c

5.0 mgl-

1BA+ 1.0

mgl-1

NAA

0 c 0 c 0 c


13 3



As showed in Table (2), the medium supplemented with

5mgl-1

BA without auxins was most effective in adventitious

shoots induction for both Sandra and Rocky cultivars. The higher

percentage of explants in Sandra cultivar that formed

organogenesis was 91.67% followed by 66.67% in Rocky. This

was significantly higher as compared with other treatments. The

average number of shoots/explant were maximum (6.5) in

Sandra cultivar (Fig.1, C) followed by 4.0 shoots/ explant in

Rocky cultivar (Fig.2, B). While the higher length of shoots

were 0.88 cm recorded in Sandra cultivar in the same medium.

2. Cotyledons organogenesis The cotyledon explants that cultured on MS medium

supplemented with different concentrations of BA combined

with different levels of kinetin or NAA (Table 3 and 4) showed

shoots regeneration which were developed after 2 weeks of

incubation, and were well developed after the end of the 6

weeks.

Table 3: Effects of BA and Kinetin combinations on direct

organogenesis from Cotyledon leaf explants of tomato

(Lycopersicon esculentum Mill.) cv. Sandra and Rocky after

six weeks of culture incubation. Tomato

cultivar

Growth

regulators

Organogenesis

%

Shoots/

explant

average

Length

average

(cm)

Sandra

0.5mgl-1

BA+ 0.5

mgl-1

Kinetin

50 3.25 b 0.69 ab

1.0 mgl-1

BA+ 0.5

mgl-1

83.34 3.71 b 1.00 a


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Kinetin

1.5 mgl-1

BA+ 0.5

mgl-1

Kinetin

100 3.50 b 0.93 a

2.0 mgl-1

BA+ 1.0

mgl-1

Kinetin

100 3.25 b 0.49 b

Rocky

0.5 mgl-1

BA+ 0.5

mgl-1

Kinetin

75 2.88 b 0.77 ab

1.0 mgl-1

BA+ 0.5

mgl-1

Kinetin

100 2.84 b 0.50 b

1.5 mgl-1

BA+ 0.5

mgl-1

Kinetin

58.33 4.58 a 0.57 b

2.0 mgl-1

BA+ 1.0

mgl-1

Kinetin

100 3.17 b 0.49 b



In Sandra cultivar, the highest organogenesis percentage

(100%) was achieved on the combination of 1.5mgl-1

BA+0.5mgl-1

Kinetin and 2.0 mgl-1

BA+1.0 mgl-1

Kinetin (Fig.1,

D). Meanwhile, the lower organogenesis percentage (50%) was

recorded in the combination of 0.5mgl-1

for each of BA and

kinetin. While in Rocky cultivar, the highest organogenesis


33 3

percentage (100%) was recorded on MS medium containing

either (1.0mgl-1

BA+0.5mgl-1

kinetin) or (2.0mgl-1

BA+1.0mgl-1

kinetin). Whereas the least organogenesis percentage (58.33%)

was observed on MS medium supplemented with (1.5 mgl-1

BA+

0.5mgl-1

kinetin), and the maximum number of shoots 4.58

/explants was recorded in this medium for cv. Rocky (Fig.2, C),

this increment was significant when compared to all other

treatments. The length of shoot reached to 1.0 cm on MS

medium supplemented with (1.0mgl-1

BA+0.5mgl-1

kinetin)

which is significantly high. In contrast, the length of shoot was

reduced drastically with increased BA and kinetin concentration,

and the least shoot length 0.49 cm attained it on MS medium

containing 2.0mgl-1

BA +1.0mgl-1

kinetin for both cultivars.

The organogenesis responses from the cotyledon leaf

explants on MS medium containing 5.0mgl-1

BA combined with

different concentrations of NAA is shown in Table 4.


NAA on direct organogenesis from Cotyledon leaf explants of


Rocky after six weeks of incubation. Tomato

cultivar

Growth

regulators

Organogenesis

%

Shoots/

explant

average

Length

average

(cm)

Sandra

5.0mgl-1

BA+ 0.0

mgl-1

NAA

33.33 1.38 b 0.25 a

5.0 mgl-1

BA+ 0.2

mgl-1

NAA

100 3.67 a 0.29 a

5.0 mgl-1

BA+ 0.5

mgl-1

NAA

0 0 e 0 c

5.0 mgl-1

16.67 0.25 c 0.08 c


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BA+ 1.0

mgl-1

NAA

Rocky

5.0 mgl-1

BA+ 0.0

mgl-1

NAA

16.67 1.0 d 0.15 b

5.0 mgl-1

BA+ 0.2

mgl-1

NAA

100 3.08 a 0.34 a

5.0 mgl-1

BA+ 0.5

mgl-1

NAA

25 0.5 c 0.13 b

5.0 mgl-1

BA+ 1.0

mgl-1

NAA

0 0 e 0 c



The highest and best shoots induction (100%) of

organogenesis percentage was noticed on MS medium

containing 5.0mgl-1


NAA.

Furthermore, the highest mean number of shoots regenerated

(3.67 and 3.08 shoots/explant) with high average of length 0.29

and 0.34 cm were observed in Sandra and Rocky respectively on

the same medium, that was significantly higher when compared

with other treatments (Fig. 1, E) and (Fig.2, D). While the

medium containing high concentration of NAA (0.5 and 1.0 mgl-

1) shows very low or no response for organogenesis in both

cultivars.

3. Leaves organogenesis Concerning the organogenesis from tomato leaf explants,

BA combinations with either NAA or kinetin were investigated

at different concentrations. Table (5) showed the organogenesis

percent of the cultured explants on MS medium supplemented

with different concentrations of growth regulators.


31 3


NAA, BA and Kinetin on direct organogenesis from leaf

explants of tomato (Lycopersicon esculentum Mill.) cv.

Sandra and Rocky after six weeks of incubation. Tomato

cultivar

Growth

regulators

Organogenesis

%

Shoots/

explant

average

Length

average

(cm)

Sandra

5.0mgl-1

BA+ 0.0

mgl-1

NAA

55.56 1.83 c 0.30 b

5.0 mgl-1

BA+ 0.2

mgl-1

NAA

22.22 1.67 c 0.10 c

0.5 mgl-1

BA+ 0.5 m

mgl-1

Kinetin

66.67 1.83 c 0.47 b

1.0 mgl-1

BA+ 0.5

mgl-1

Kinetin

77.78 2.22 bc 0.24 b

Rocky

5.0 mgl-1

BA+ 0.0

mgl-1

NAA

44.44 3.83 a 0.18 b

5.0 mgl-1

BA+ 0.2

mgl-1

NAA

0.0 0.0 d 0.0 c

0.5 mgl-1

BA+ 0.5

mgl-1

Kinetin

33.33 2.67 b 0.63 a

1.0mg/l

BA+0.5 mg/l

Kinetin

66.67 2.67 b 0.60 a


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The highest organogenesis percentages (77.78 and

66.67%) were achieved from both cultivars (Sandra and Rocky)

respectively on the combination (1.0 mgl-1

BA+0.5mgl-1

kinetin)

which were higher than the other treatments. Whereas, the least

organogenesis percentage (22.22%) was found on the medium

augmented with 5.0mgl-1

BA+ 0.2mgl-1

NAA for Sandra

cultivar, while in Rocky cultivar this medium did not show any

response for organogenesis percentage.

The number of shoots/explant increased from 2.67 to 3.83

shoots/explant by changing the combination from media

containing (1.0 mgl-1

BA+0.5mgl-1

kinetin) to MS medium


BA in Rocky cultivar (Fig.2, E).

This increase was highly significant when compared to all other

treatments. Regarding Sandra cultivar, the highest number of

shoots/explant 2.22 was recorded on MS medium supplemented

with 1.0 mgl-1

BA combinations with 0.5 mgl-1

kinetin.

The minimum shoot length was attained on MS medium



NAA,

which reached to 0.10 cm as an average in Sandra cv., while the

highest shoot length reached to 0.63 cm on MS medium

supplemented with 0.5mgl-1

BA+0.5mgl-1

kinetin in Rocky

cultivar, that is significantly high when compared to all other

treatments.

The regenerated shoots were rooted on MS medium and then

transferred to pots containing soil in the greenhouse (Fig.1, F)

and (Fig.2, F).

Discussion

The results in table 2 which related to the effect of the

combination between BA and Kinetin are in agreement with

previous findings that BA engorges direct shoot regeneration and


33 3

the production of shoots per explant from hypocotyls of tomato

when used in combination with kinetin (Rashid and Bal, 2010).

Moreover, Hnur and Krishnareddy (2016) clearly indicated the

role of kinetin with BAP into produce good embryogenic calli

from hypocotyls explants in tomato and developed them to

whole plantlets. Furthermore, many researchers have reported

that kinetin and BA as important cytokinins with high shoot

regeneration ability from hypocotyl explants when used in

combination with some auxins such as NAA in the culture

medium (Manawadu et al., 2014). Kauser et al. (2016) as well

showed that the combination of 1.5mgl-1

BAP, 0.5mgl-1

zeatin

and 0.2mgl-1

IAA was considered as the best medium for high

plant regeneration rates from hypocotyl, cotyledon and leaf

explants. These results can be explained that the effect of two

types of cytokinins is more influence on direct shoot

regeneration in tomato when compared with using one cytokinin

(Kauser et al., 2016). However, other researchers found that the

best regeneration medium for hypocotyls explants with high

regeneration frequency 100% was MS medium supplemented

with 1 mgl-1

zeatin and 0.1 mgl-1

of indole 3- acetic acid (Gubis

et al., 2004). Gubis et al. (2003) studied the regeneration ability

of six types of explants in 13 cultivars of tomato and found that

the most responsive explants were hypocotyles and epicotyls

with up to 100% regeneration with average number of shoots

range 6.3 and 6.5/explant respectively.

The findings in table 2 are in agreement with those

published by Mohamed et al. (2010) who found that the MS

medium supplemented with 2.0 mgl-1

BAP without any auxin

induced high organogenesis percentage, high shoot number/

explant and high shoot length from hypocotyl explants of

tomato. However, other studies have reported that MS medium

supplemented with BAP and IAA was the best medium for shoot


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regeneration from hypocotyl explants of tomato (Wayase and

Shitole 2014; Rashid et al., 2012). The reason behind the

superiority of BAP alone on the BAP combined with NAA in

shoot organogenesis from hypocotyl explants is due to the role

of BAP in cell division and auxiliary bud formation (Sutter,

1996), as well as the ability to regeneration was substantially

dependent on the type of explants (Gerszberg et al., 2016).

The results of table 3 are corresponded with other

researches that demonstrate the role of cytokinin on In vitro

organogenesis by using kinetin combination with BAP that

hence callus induction and shoot regeneration from cotyledon

leaves of tomato plant in absence of auxin (Rashid and Bal,

2010). In addition, Hnur and Krishnareddy (2016) also noted that

the callus induction and plant regeneration from cotyledonous

leaves of tomato plant was greatly influenced by addition of

kinetin and BAP to MS medium. As well as, the reduction of

organogenesis percentage from cotyledon leaf explants

comparing with hypocotyl explants on the same medium may be

due to the type of explant which used in the experiment.

Similar results of table 4 were obtained by Kalyani and

Rao (2014) who found an efficient protocol for direct shoot

regeneration from cotyledon explants of tomato, using MS

medium supplemented with 1.0 mgl-1

zeatin and 0.1mgl-1

IAA.

Other studies have reported that the best explants for inducing

shoot regeneration were cotyledon explants followed by

hypocotyls of tomato on MS medium supplemented with 2.0mgl-

1 BA and 0.1mgl

-1 IAA (Gerszberg et al., 2016). Wayase and

Shitole (2014) showed that the best medium for highest number

of shoot induction from cotyledon explants (7.8 shoots /

explant) were produce on MS medium containing 6.65 µM BAP

combined with 1.14 µM IAA. The reasons behind the positive

role of the combination between the cytokinins and the auxins on


33 3

direct organogenesis in tomato might be due to the morphogenic

response by modifying various physiological processes (Wayase

and shitole, 2014).

The role of various concentrations of auxins and cytokinin

in direct shoot regeneration or organogenesis of tomato was

documented by Manawadu et al., (2014). As well as, Kauser et

al., (2016) studied the effect of various combination between

BAP, zeatin , IAA and IBA on direct shoot regeneration from

leaf explants. While other studies found that the direct shoot

regeneration obtained from leaf explants of cv. Moneymaker on

a medium containing 1.0 mgl-1

zeatin, 1.0 mg/l IAA with B5

vitamins (Sarwarkhan et al., 2006). The reason of lower

organogenesis percentage and shoots number from leaf explants

comparing with cotyledon and hypocotyl explants in this study

may be due to the type of explants as well as the type of plant

growth regulators and concentration (Majoul et al., 2007).

However, other research found that the better explants for direct

regeneration was the leaf bud when cultured on MS medium


kinetin +2.0 mgl-1

BAP + 0.1 mgl-1

NAA (Manawadu et al., 2014).

Figure 1: Direct organogenesis in tomato cv. Sandra

A. The twelve days old seedling of in vitro grown tomato.

B. Shoots differentiation from hypocotyls explants on MS

medium augmented with 1.0 mgl-1

BA+ 0.5mgl-1

kinetin after

6 weeks of culture.

C. Shoots differentiation from hypocotyl explants on MS

medium augmented with 5.0mgl-1

BA+ 1.0mgl-1

NAA after 6

weeks of culture.

D. Shoots differentiation from cotyledon explants on MS


BA+ 0.2mgl-1

NAA after 6

weeks of culture.


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E. Shoots differentiation from cotyledon explants on MS


BA+ 1.0mgl-1

kinetin after 6

weeks of culture.


33 3

F. Plantlets transferred to the soil in the greenhouse.

Figure 2: Direct organogenesis in tomato cv. Rocky

A. Shoots differentiation from hypocotyls explants on MS


BA+ 0.5mgl-1

kinetin after

4 weeks of culture.

B. Shoots differentiation from hypocotyl explants on MS

medium augmented with 1.0 mgl-1

BA+ 0.5mgl-1

kinetin after

6 weeks of culture.

C. Shoots differentiation from cotyledon explants on MS


BA+ 0.2mgl-1

NAA after 4

weeks of culture.

D. Shoots differentiation from cotyledon explants on MS


BA+ 0.2mgl-1

NAA after 6

weeks of culture.

A B C

D E F


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E. Shoots differentiation from leaf explants on MS medium

augmented with 1.0 mgl-1

BA+ 0.5mgl-1

kinetin after 6 weeks of

culture.

F. Plantlets transferred to the soil in the greenhouse.

Conclusion

In present study an efficient protocol for direct

organogenesis from hypocotyls and cotyledon leaves in tomato

plants through multiple shoot induction has been standardized,

the regenerated plants were transferred to the soil successfully.

The protocol established here can be serving as an efficient

method for the rapid propagation and conservation of genetic

transformations and many other biotechnological aspects for

future studies.


33 3

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Documents

المجلــــد (2) ، العـــــدد (6)،...Table (1) showed that the highest organogenesis percentage (100% ) was found on MS medium augmented with 0.5 and 1.0mgl-1 BA